Not applicable.
1. Field of the Invention
This invention relates to fluorescent compounds useful as indicator molecules for detecting the presence or concentration of an analyte in a medium, such as a liquid, and to methods for achieving such detection. More particularly, the invention relates to fluorescent lanthanide metal chelate complexes containing substituted ligands and their use as indicator molecules for detecting the presence or concentration of an analyte such as glucose or other cis-diol compound in a medium, including a liquid medium such as a biological fluid.
2. Description of the Related Art
Certain rare-earth metal chelates emit visible light upon irradiation with UV light and different forms of visible light (e.g., violet or blue light), an emission which is characterized by the chelated cation. Some lanthanide ions, such as those of europium (Eu3+), samarium (Sm3+), terbium (Tb3+), and to a lesser extent dysprosium (Dy3+) and neodymium (Nd3+), exhibit typical fluorescence characterized by the ion, especially when chelated to suitable excitation energy mediating organic ligands. The fluorescent properties of these compoundsxe2x80x94long Stokes"" shift, narrow band-type emission lines, and unusually long fluorescence lifetimesxe2x80x94have made them attractive candidates for fluorescent immunoassays and time-resolved fluorometric techniques.
The major emission lines of these fluorescent lanthanide chelates are formed from a transition called hypersensitive transition and are around 613-615 nm with Eu3+, 545 (and 490) nm with Tb3+, 590 and 643 nm with Sm3+, and 573 with Dy3+. See Hemmila, Application of Fluorescence in Immunoassays, 140-42 (1991). See also Spectroscopy in Inorganic Chemistry, vol. 2, at 255-85 (Academic Press 1971). Radiation is typically absorbed by the chelates at a wavelength characteristic of the organic ligand and emitted as a line spectrum characteristic of the metal ion because of an intramolecular energy transfer from the ligand to the central metal ion. The organic ligand absorbs energy and is raised or excited from its singlet ground state, S0, to any one of the vibrational multiplets of the first singlet excited state, S1, where it rapidly loses its excess vibrational energy. At this point, there are two possibilities: relaxation by an S1xe2x86x92S0 transition (ligand fluorescence) or intersystem crossing to one of the triplet states, T1. See E. P. Diamandis et al., Analytical Chemistry 62:(22):1149A (1990); see also Spectroscopy in Inorganic Chemistry, vol. 2, at 255-85 (Academic Press 1971).
Fluorescent europium chelates are known to exhibit large Stokes shifts (xcx9c290 nm) with no overlap between the excitation and emission spectra and very narrow (10-nm bandwidth) emission spectra at 615 nm. In addition, the long fluorescence lifetimes (measurable in microseconds instead of the nanosecond lifetimes measurable for conventional fluorophores) of the chelates help filter out noise and other interference having a low fluorescent lifetime. The long fluorescent lifetimes thus permit use of the chelates for microsecond time-resolved fluorescence measurements, which further reduce the observed background signals. Additional advantages of using europium chelates include that europium chelates are not quenched by oxygen.
Line emissions of two europium (Eu) chelates, Eu-dibenzoylmethide and Eu-benzoylacetonate, have made the chelates attractive candidates for use in lasers. See H. Samuelson, et al. (J. Chem. Physics 39(1): 110-12 (1963)) Samuelson, et al. studied the fluorescence and absorption of the above two europium chelates as solids and in solution. Samuelson, et al. compared the fluorescent lifetimes of the europium chelates under various conditions with the lifetimes of europium fluorescence in other compounds. Based on this comparison, Samuelson, et al. suggested that the variation in lifetimes between the two groups of europium compounds is a result of the ligand-Eu interaction in the europium chelates. Specifically, Samuelson et al. determined that various emission lines from Eu-dibenzoylmethide showed fluorescent lifetimes of 480 +/xe2x88x9250 xcexcs, which were significantly greater than the fluorescent lifetimes in other europium compounds.
Crosby, et al., J. Chem. Physics 34:743 (1961) had previously studied the role of intramolecular energy transfer in sensitizing ion emission from rare-earth metal chelates, including europium dibenzoylmethide and europium benzoylacetonate chelates. Whan, et al., J. Mol. Spectroscopy 8: 315-27 (1962) reported that the emission from chelates of a group of lanthanide metal ions (Eu3+, Tb3+, Dy3+ and Sm3+) was dominated by bright spectral lines characteristic of the individual rare-earth metal ions. Whan, et al. found that both the benzoylacetonates and dibenzoylmethides of Eu3+ and Tb3+ are especially bright emitters and that the bright line emissions and low yields of phosphorescence from these chelates indicated that intramolecular energy transfer from the ligands to the Eu3+ and Tb3+ ions of these chelates occurs efficiently. Whan, et al., at 324.
N. Filipescu, et al., J. Physical Chem. 68(11):3324 (1964) reported that the fluorescence spectra of europium and terbium xcex2-diketone chelates are modified when substituents are changed in the organic ligand portion of the chelates. Filipescu, et al. discussed the relative intensity, spectral distribution, shifting, and splitting of the fluorescence lines of the europium and terbium chelates in relation to the nature of substituents, their position, molecular configuration, and the overall intramolecular energy transfer. Filipescu, et al. found that the overall fluorescence intensity characteristic of the ion depended on two factors: 1) the amount of energy available at the organic triplet, and 2) the efficiency of energy transfer to the ion.
Filipescu, et al. also found that the above two factors varied for different substituents. For instance, the substitution of europium dibenzoylmethide chelates with electron-donor methoxy groups in the meta position on the chelate was found to enhance the fluorescent emission of the europium ion, whereas paramethoxy substitution was found to decrease the europium fluorescence. Additionally, the effect was more pronounced for the di- than for the monomethoxy-substituted dibenzoylmethides. In contrast, an opposite effect was observed for nitro-substituted dibenzoylmethides of europium. The electron-withdrawing nitro groups attached to the para or meta positions were found to decrease the total ionic emission of europium. Additionally, the effect was more pronounced for di- than for monosubstituted dibenzoylmethides.
Filipescu, et al. further found that the strong ionic fluorescence emitted by europium para-phenyldibenzoylmethide indicated that increasing the size of the aromatic system enhanced the amount of energy transferred to the europium ion. This fact was confirmed by the emission results obtained for napthyl-substituted diketones which were found to have substantially higher ionic emissions than the dibenzoylmethide chelates. Filipescu, et al., at 3328-29.
E. Diamandis, et al., Analytical Chemistry 62(22): 1149A (1990), described how europium chelates can be used as labels in fluorescence immunoassays and DNA hybridization assays. With respect to fluorescent immunoasays, the authors described that europium chelates can be used as immunological labels in various assay configurations, including either competitive or noncompetitive assays.
U.S. Pat. No. 4,374,120 (Soini, et al.) describes a method for detecting a substance using a fluorescent lanthanide chelate complex as a marker. U.S. Pat. No. 4,374,120 also describes the use of xcex2-diketones as enhancing ligands for promoting the strong fluorescence properties of certain lanthanide chelates, especially chelates of europium and terbium.
Wallac (Turku, Finland) developed a lanthanide metal chelate to replace radiation tags for conducting immunoassays, having the structure: 
The Wallac molecule was found to behave very efficiently in dilute solutions. See Hemmila, Applications of Fluorescence in Immunoassays, p. 149 (1991).
Certain conditions are required for using lanthanide metal chelates in aqueous solutions, such as in biological fluids. For example, it is known that chelates must, first, be dissolved in the aqueous solution, and second, avoid being quenched by water molecules which tend to fill up the empty coordination sites of the lanthanide ion. However, various adducts or Lewis bases, such as phosphines, phosphine oxides, or nitrogen heterocycles, have been used in addition to the ligand structure to form an xe2x80x9cinsulating sheetxe2x80x9d around the lanthanide ion, enhancing the fluorescence by preventing water molecules from penetrating into the complex""s inner sphere. For example, solutions developed for fluorometric determinations of lanthanides in aqueous systems (e.g., immunoassays) have comprised xcex2-diketones and trioctylphosphine oxide (xe2x80x9cTOPOxe2x80x9d) as an adduct forming synergistic agent, and a detergent (e.g., Triton X100) which forms micelles and helps to solubilize the coordinated complex. See Applications of Fluorescence in Immunoassays, at 146-47.
Lanthanide metal chelate complexes have not been previously examined or constructed for the purpose of active detection of an analyte by utilizing a discrete and specific recognition element feature, such as a boronate group for detecting glucose and other cis-diols, through one or more ligands contained in the chelate complex. As discussed above, lanthanide metal chelates have been investigated primarily for use as laser dyes, substitute labels for radioisotopes, and for attachment to antibodies as labels in immunoassays. Lanthanide metal chelates also have been used for qualitative analytical procedures for detecting tetracycline.
Glucose is an organic compound indispensable to living organisms and plays an important role in information transmission, energy metabolism and structure formation in such organisms. For example, glucose, and more particularly, D-glucose, is crucial as an energy source for a variety of cells in constructing various organs. Glucose is stored in the liver as glycogen, which is released in body fluids as needed for energy consumption. The production and consumption of glucose are well balanced in the body fluids of a normal or healthy human being, maintaining the glucose concentration constant in the fluids. Thus, detecting sub-levels or supra-levels of glucose in the blood or the urine provides valuable information for diagnosing such diseases as diabetes and adrenal insufficiency.
A glucose sensor using an enzyme (e.g., as made by Yellow Springs Instruments (YSI), Ohio) is the best known practical measure for detecting glucose. This technique involves decomposing glucose with an enzyme (glucose oxidase) and measuring the amount of hydrogen peroxide produced by the decomposition through an appropriate means (such as by an electrode). Although this method is well established, the quality of the enzyme, which originates from a living body, will irreversibly change over time and cannot be recycled for reuse. Additionally, because the glucose is actually consumed in the detection reaction, the intrinsic ability of the glucose sensor to measure low levels of analyte is therefore limited.
It is well known that boronic acid-containing compounds bind to glucose. The mechanism is believed to occur through bonding of adjacent hydroxyl groups on glucose to hydroxyl groups on a boronate moiety, as drawn below: 
The complexation of carbohydrates, including glucose, with phenylboronic acid has been known for a long time and the reversibility of that interaction has served as a basis for the chromatographic separation of sugars. Specifically, in 1959, Lorand and Edwards reported association constants for aqueous associations of phenylboronic acid with many saturated polyols; binding interactions ranged from very weak (e.g., ethylene glycol, Kd=360 mM) to moderately strong (e.g., glucose, Kd=9.1 mM). See J. Yoon, et al., Bioorganic and Medicinal Chemistry 1(4): 267-71 (1993).
U.S. Pat. No. 5,503,770 (James, et al.) describes a fluorescent boronic acid-containing compound that emits fluorescence of a high intensity upon binding to saccharides, including glucose. The fluorescent compound has a molecular structure comprising a fluorophore, at least one phenylboronic acid moiety and at least one amine-providing nitrogen atom where the nitrogen atom is disposed in the vicinity of the phenylboronic acid moiety so as to interact intermolecularly with the boronic acid. Such interaction thereby causes the compound to emit fluorescence upon saccharide binding. U.S. Pat. No. 5,503,770 describes the compound as suitable for detecting saccharides. See also T. James, et al., J. Am. Chem. Soc. 117(35): 8982-87 (1995).
Additionally, fluorescent sensors using an anthrylboronic acid-containing compound for detecting blood glucose are known in the art. For example, J. Yoon, et al., J. Am. Chem. Soc. 114:5874-5875 (1992) describe that anthrylboronic acid can be used as a fluorescent chemosensor for signaling carbohydrate binding, including binding of glucose and fructose.
An object of the present invention is to detect the presence or concentration of an analyte in a medium such as a liquid or gas by measuring any change in fluorescence emitted by a lanthanide metal chelate complex upon binding of the analyte to one or more chelators of the chelate complex through an analyte-specific recognition element.
Another object of the present invention is to provide an analyte-specific, recognition element-containing lanthanide metal chelate complex as an indicator molecule for detecting the presence or concentration of an analyte such as glucose or other cis-diol compound in a medium such as a liquid.
The present invention is directed to an indicator molecule for detecting the presence or concentration of an analyte, comprising a fluorescent lanthanide metal chelate complex having the formula:
M(xe2x80x94Ch (xe2x80x94RX))Y
wherein:
M represents a lanthanide metal ion; Ch represents a chelator comprising a ligand, preferably an organic ligand which can comprise any one or more of a xcex2-diketone or a nitrogen analog thereof, a dihydroxy, a carboxyl coordinating heterocycle, an enol, a macrobicyclic cryptand (i.e., a cage-type ligand), a phenylphosphonic acid, or a polyamino-polycarboxylic acid. The organic ligand of Ch can also comprise any one or more of a heterocycle of nitrogen, sulfur, and linked carboxyls. The organic ligand of Ch can further comprise any one or more of an alkane or alkene group, preferably containing 1 to 10 carbon atoms, as well as aromatic, carbocyclic or heterocyclic moieties, including benzyl, napthyl, anthryl, phenanthryl, or tetracyl groups. Furthermore, one or more chelators complexed with M can be the same or a mixture of different chelators (so-called xe2x80x9cmixed ligand or ternary chelatesxe2x80x9d).
R represents an analyte-specific recognition element, one or more of which is bound to one or more ligands of the chelate complex, but need not be linked to every ligand of the chelate complex. In a preferred embodiment of the present invention, R can be a boronate group or a compound containing a boronate group for detecting glucose or other cis-diol compound.
X represents the number of recognition elements R bound to each of one or more chelators. X can be an integer from 0 to 8, and in certain preferred embodiments of the invention, X=0 to 4 or X=0 to 2. Additionally, the number of recognition elements R bound to each of one or more chelators may be the same or different, provided that for one or more chelators, X greater than 0. Y represents the number of chelators complexed with M, and can be an integer from 1 to 4. In certain preferred embodiments of the invention, Y=1, Y=3 or Y=4.
The present invention also is directed to a fluorescent lanthanide metal chelate complex, as defined above.
The present invention further is directed to methods for detecting the presence or concentration of an analyte by utilizing the above indicator molecule and fluorescent lanthanide metal chelate complex. The method comprises the steps of exposing the sample to an indicator molecule comprising a fluorescent lanthanide metal chelate complex having the above-defined formula, and measuring any change in fluorescence emitted by the lanthanide metal chelate complex, and therey detecting the presence or concentration of the analyte.
In the present invention, the presence or concentration of the analyte is detected by measuring any change in fluorescence emitted by the lanthanide metal chelate complex upon binding of the analyte to one or more chelators of the chelate complex through one or more analyte-specific recognition elements. Specifically, the presence or concentration of an analyte, such as glucose or other cis-diol compound, is determined by observing and/or measuring the change in intensity or lifetime of fluorescence emitted by the fluorescent metal ion (i.e., the fluorescence is attenuated, enhanced or shifted in wavelength) upon binding of the analyte to the analyte-specific recognition element of the chelate, which for detecting glucose or other cis-diol compound is a boronate-containing recognition element.
The present invention offers the advantage of being able to detect an analyte, such as glucose or other cis-diol compound, in an analyte-specific manner in a medium such as a liquid or a gas, utilizing a fluorescent indicator molecule having a fluorescent lifetime of sufficient length (measurable in microseconds instead of nanoseconds), as well as having a long Stoke""s shift, thereby decreasing the effect of any background noise and other interference which would reduce the sensitivity of the analyte detection, and is not concentration quenched.